In the mitogen-activated protein kinase (MAPK) cell signaling system, MKKKs phosphorylate and activate MKKs, which in turn phosphorylate and activate MAPKs (JNK, ERK1/2, p38, and ERK5). For example, the MKKK MEKK2 regulates the JNK signaling pathway, whereas the closely related MEKK3 controls p38 activity, thus establishing distinct cellular functions. However, both MEKK2 and MEKK3 regulate a third MAPK pathway resulting in the activation of ERK5, and consequently regulate ERK5-dependent gene expression. The MEKK2/3-MEK5 pathway is the predominant MAPK module to control ERK5 activation. ERK5 is activated by a variety of growth factors and stress stimuli, and genetic approaches have demonstrated that ERK5 is involved in angiogenesis and cardiac hypertrophy. MEKK2/3 and MEK5 interact by their N-terminal PB1 (Phox/Bem1p) domains that are unique among known MAPK module components, and this interaction regulates activation of MEK5 ERK5, but not JNK or p38. Importantly, while the signals that influence MAPK activation can be diverse, ERK5 signaling converges at the MEKK2/3-MEK5 nexus; a function mediated by PB1 domain interaction. This essential interaction therefore presents a unique opportunity to specifically block ERK5 signaling. We propose to develop an assay for ERK5 pathway signaling based on this PB1 domain interaction that will facilitate high throughput screening of potential ERK5 signaling inhibitors that function through disrupting PB1 domain association. Targeting MEKK2/3- MEK5 interaction by small molecule inhibitors will markedly disrupt ERK5 activation and selectively inhibit stimulus-specific activation of cytokine expression in multiple cell types, and thus provide new opportunities for therapeutic intervention in diseases involving inflammation.